C
HARACTERIZATION OF
S
OME
E
ARLY
D
EFENCE
R
ESPONSES OF
L
EAF
R
UST
‐I
NFECTED
W
HEAT
by
J
OHANNES
J
ACOBUS
A
PPELGRYN
Submitted in fulfilment of the requirements for the degree
P
HILOSOPHIAE
D
OCTOR
in the Faculty of Natural and Agricultural Sciences
Department of Plant Sciences
University of the Free State
Bloemfontein
South Africa
2007
Promoter:
Dr B Visser
Co‐Promoter:
Prof ZA Pretorius
I prefer the errors of enthusiasm to the indifference of wisdom – Anatole
France
To succeed….. you need to find something to hold on to, something to
motivate you, something to inspire you – Tony Dorsett
I am greatly indebted by the following people:
• My wife, Anneke, for all your support and understanding without which this would not have been possible.
• Dr Botma Visser, promoter, thank you for your guidance and support throughout this study.
• Prof Sakkie Pretorius, co‐promotor, thank you for your input to make this study a success. Thank you also for making the glasshouse space, seeds and rust spores available. • Cornel Bender, for your technical help with the microscopy and help and guidance with the rust inoculations. • My parents and parents‐in‐law, thank you for your support as well. • My friends and colleagues in the lab and the Dept of Plant Sciences, thank you for your help and friendship. I am greatly indebted to the following institutions: • The Department of Plant Sciences and the University of the Free State, for providing the facilities and resources necessary to complete this study • The NRF for financial support.
TABLES AND FIGURES IX
CHAPTER 1: INTRODUCTION 1
CHAPTER 2: LITERATURE REVIEW 5
2.1 INTRODUCTION 6
2.2 PLANT‐PATHOGEN INTERACTION 6
2.2.1 Wheat Leaf Rust 7 2.3 PATHOGEN RECOGNITION 8 2.4 DEFENSE RESPONSES 9 2.4.1 Systemic Resistance 9 2.4.2 Programmed Cell Death 10 2.4.3 H2O2 11 2.4.4 Salicylic Acid 12 2.4.5 Jasmonic Acid 12 2.4.6 Ethylene 13
2.5 CHANGES IN GENE ACTIVITY 14
2.5.1 Molecular Chaperones 14 2.5.2 Transcription factors 17 2.5.3 R‐genes 18 2.6 INTERPLANT COMMUNICATION 20 2.7 CONCLUDING REMARKS 23 CHAPTER 3: CLONING OF DIFFERENTIALLY EXPRESSED CDNA FRAGMENTS FROM WHEAT 25 3.1 INTRODUCTION 26 3.2 MATERIALS AND METHODS 27 3.2.1 Plant Material 27 3.2.2 Leaf Rust Inoculation 27 3.2.3 Differential Display Reverse Transcription Polymerase Chain Reaction (DDRT‐PCR) 28 3.2.4 Reverse Northern Blot Analysis 31 3.2.5 Sequencing of Cloned cDNA Fragments 31 3.3 RESULTS 31 3.3.1 Leaf Rust Infection 31 3.3.2 Identification of Differentially Expressed Genes 31 3.4 DISCUSSION 39 3.5 REFERENCES 43
4.2.1 Biological Material 49 4.2.2 Rust Inoculation Of Wheat 50 4.2.3 Differential Display of Leaf Rust Infected Wheat RNA 50 4.2.4 Sequencing and DNA Analysis 51 4.2.5 Southern and Northern Blot Analysis 51 4.2.6 Expression Analysis Using RT‐PCR 52 4.2.7 Western Blot Analysis 52 4.2.8 Chemical Treatments of Wheat 53 4.3 RESULTS 53 4.3.1 Identification of TaHlp01 53 4.3.2 Genomic Presence of TaHlp01 60 4.3.3 Expression Analysis of TaHlp01 60 4.3.4 Chemical Induction of TaHlp01 Expression 66 4.4 DISCUSSION 69 4.5 REFERENCES 76 CHAPTER 5: EVIDENCE FOR VOLATILE DEFENCE 82 5.1 INTRODUCTION 83 5.2 MATERIALS AND METHODS 84 5.2.1 Biological Material 84 5.2.2 Leaf Rust Inoculation Of Wheat 84 5.2.3 An Investigation Into Possible Plant Communication 85 5.2.4 Western Blot Analysis 85 5.2.5 ‐1,3‐glucanase Activity Determination 86 5.2.6 RT‐PCR Analysis 86 5.2.7 Fluorescence Microscopy 87 5.3 RESULTS 87 5.3.1 Thatcher Infection‐Related Communication 88 5.3.2 Thatcher+Lr34 Infection‐Related Communication 93 5.3.3 Phenotypic Analysis Of The Defense Response Of US And UR Plants 98 5.4 DISCUSSION 100 5.5 REFERENCES 107 CHAPTER 6: GENERAL DISCUSSION 113 REFERENCES 121
OPSOMMING 158
Abbreviations
A APX Ascorbate peroxidases AVG Aminoethoxyvinylglycine AVR Avirulence B BTH Benzo (1,2,3) thiadiazole‐7‐carbothioic acid S‐methyl ester bZIP Basic‐domain leucine zipper C CAT Catalase CC Coiled‐coil CWA Cell wall appositions CR Control resistant CS Control susceptible D DDRT‐PCR Differential display reverse transcription polymerase chain reaction dCTP Deoxycytosine triphosphate DIBOA 2,4‐dihydroxy‐2H‐1,4‐benzoxazin‐3(4H)‐one DIMBOA 2,4‐dihydroxy‐7‐methoxy‐2H‐1,4‐benzoxazin‐3(4H)‐one dNTP’s Deoxynucleotide triphosphates DTT Dithiothreitol E ERF Ethylene‐responsive‐element‐binding factors G GLV Green leaf volatiles H
HR Hypersensitive response HSF Heat‐shock factors Hsp Heat‐shock proteins I IR Infected resistant IS Infected susceptible ISR Induced systemic resistance J JA Jasmonic acid L LAR Localized acquired resistance LIR Later infected resistant LIS Later infected susceptible LOX Lipoxygenase Lr Leaf rust resistance LRR Leucine‐rich repeats M MeJA Methyl jasmonate MeSA Methyl salicylate N NBS Nucleotide binding site NO Nitric oxide NPR1 Non‐expressor of PR1
PAL Phenylalanine ammonia‐lyase PBD Peptide‐binding domain PCD Programmed cell death PR Pathogenesis related PAHBAH p‐Hydroxybenzoic acid hydrazide R R Resistance genes RLK Receptor‐like protein kinases RNI Reactive nitrogen intermediates ROI Reactive oxygen intermediates ROS Reactive oxygen species RT Reverse transcription S SA Salicylic acid SAR Systemic acquired resistance sHsp Small Heat‐shock proteins SOD Superoxide dismutase T TaHlp01 Triticum aestivum Heat shock‐like protein 1 TIR Toll/Interleukin‐1 receptor TMV Tobacco mosaic virus Tween™ 20 Polyoxyethylene sorbitan monolaurate U UR Uninfected resistant US Uninfected susceptible
Tables and Figures
Table 3.1: Names and sequences of the primers used during Differential Display 29 Figure 3.1: The amplification strategy used for the DDRT‐PCR. 30 Figure 3.2: Flag leaves of adult Thatcher and Thatcher+LR34 plants one week after
infection with P. triticina
32
Figure 3.3: Reverse Northern blot analysis of cloned cDNA fragments 34
Figure 3.4: Sequence analysis of clone M8 35
Figure 3.5: Sequence analysis of clone M9 36
Figure 3.6: Sequence analysis of clone M27 37
Figure 3.7: Sequence analysis of clone D5 39
Figure 4.1: RT‐PCR amplification of TaHlp01 and TaHsp70h 52
Figure 4.2: Sequence analysis of TaHlp01 54
Figure 4.3: Amino acid sequence alignment of Contig 18805 and TaHsp70h 55
Figure 4.4: Sequence analysis of TaHsp70h 56
Figure 4.5: Amino acid sequence alignment of TaHlp01 and TaHsp70h 58 Figure 4.6: Southern blot analysis of Thatcher and Thatcher+Lr34 genomic DNA
probed with TaHlp01
59
Figure 4.7: Southern blot analysis of different leaf rust resistant wheat cultivars probed with TaHlp01
60
Figure 4.8: Expression analysis of TaHlp01 and TaHsp70h in P. triticina infected Thatcher and Thatcher+Lr34 wheat
61
Figure 4.9: Immunological detection of expressed polypeptides 63 Figure 4.10: Expression analysis of TaHlp01 in P. striiformis infected Avoset wheat 64 Figure 4.11: Expression analysis of TaHlp01 during the treatment of Thatcher+Lr34
with different chemicals
Figure 5.1: Selected defence related marker gene expression during the interaction between infected Thatcher (IS) and uninfected Thatcher (US) and Thatcher+Lr34 (UR)
87
Figure 5.2: β‐1,3‐glucanase activity for infected Thatcher (IS) and its interaction with Thatcher (US) and Thatcher+Lr34 (UR)
88
Figure 5.3: Expression of marker genes involved in different volatile signalling pathways during the interaction between infected Thatcher (IS) and uninfected Thatcher (US) and uninfected Thatcher+Lr34 (UR)
89
Figure 5.4: Selected defence related marker gene expression during the interaction between infected Thatcher+Lr34 (IR) and uninfected Thatcher (US) and Thatcher+Lr34 (UR)
92
Figure 5.5: β‐1,3‐glucanase activity for infected Thatcher+Lr34 (IR) and its interaction with uninfected Thatcher (US) and Thatcher+Lr34 (UR)
93
Figure 5.6: Expression of indicator genes involved in different volatile signalling pathways during the interaction between infected Thatcher (IS) and uninfected Thatcher (US) and uninfected Thatcher+Lr34 (UR)
94
Figure 5.7: The effect of volatile emissions on the defence response of US and UR wheat
96
Figure 5.8: Visible effect of volatile emissions on the defence responses of infected wheat 98
Plants are exploited as a source of food and shelter by a wide range of organisms, including viruses, bacteria, fungi, nematodes, insects and even other plants (Odjakova and Hadjiivanova, 2001). The identification of potential pathogenic microbes by the plant leads to the activation of different defence responses which are designed to prevent further infection (Thatcher et al., 2005). Disease resistance in plants is usually associated with the activation of a wide variety of defence responses that serve to prevent pathogen replication and/or movement.
In some plant‐pathogen interactions, the ability of the host plant to recognize the pathogen and activate these responses is regulated in a gene‐for‐gene‐specific manner by the direct or indirect interaction between the products of a plant disease resistance (R) gene and a pathogenic avirulence (Avr) gene (Flor, 1971; Marois et al., 2002; Axtell and Staskawicz, 2003; Di Gaspero and Cipriani, 2003). When either the plant or the pathogen lacks its cognate gene, activation of the plant’s defence responses either fails to occur or is delayed sufficiently so that pathogen colonization ensues (Flor, 1956; Dangl and McDowell, 2006). In contrast to this race/cultivar‐specific form of resistance which is relatively rare, many plant species exhibit non‐host resistance. Non‐host resistance is characterized by the activation of many of the same defence responses as are associated with race/cultivar‐specific resistance. However, it occurs in the absence of any known R/Avr gene combination (Heath 2001;
Thordal‐Christensen, 2003; Dodds et al., 2006).
Resistance in plants is manifested by the inability of the pathogen to grow or multiply and spread and often takes the form of a hypersensitive response (HR) (Vranová, 2002). The hypersensitive response is characterized by localized cell death at the site of infection (Van Loon, 1997). As a result, the pathogen remains confined to necrotic lesions near the site of infection. A ring of cells surrounding the necrotic lesion becomes refractory to subsequent infection. This is known as localized acquired resistance (LAR) (Fritig et al., 1998; Ghannam et al., 2005). These local responses trigger specific and nonspecific resistance throughout the plant which is known as systemic acquired resistance (SAR) and provides durable protection against infection by a broad range of pathogens (Scheel, 1998; Durrant and Dong, 2004).
a non‐host specific elicitor is the oxidative burst, in which levels of reactive oxygen species (ROS) rapidly increase (Mittler et al., 2004; Neill et al., 2002a). Other rapid responses include the crosslinking of cell wall proteins (McLusky et al., 1999), the activation of protein kinases (Romeis, 2001) and the increased expression of various defence genes. Some of these genes encode peroxidases, glutathione S‐transferases, proteinase inhibitors and various biosynthetic enzymes such as phenylalanine ammonia lyase (PAL) and pathogenesis‐ related (PR) proteins (Kessmann et al., 1994; Klessig et al., 2000; Flors and Nonell, 2006). Activation of signal transduction networks after pathogen recognition results in the reprogramming of cellular metabolism, involving large changes in gene transcriptional activity while basic incompatibility frequently results in the expression of defence related genes and localized host cell death (Yamamoto et al., 2004).
Plants contain many defence related proteins. In addition to R‐genes and genes encoding signal transduction proteins, they also possess downstream defence genes (Van Loon, 1997), enzymes involved in the generation of phytoalexins (Flors and Nonell, 2006), enzymes involved in oxidative stress protection (May et al., 1998), lignification (Cano‐ Delgado et al., 2003) and numerous others. Many of these genes are involved in the production of secondary metabolites such as those of the shikimate (Herrmann and Weaver, 1999) and phenylpropanoid pathways (Dixon et al., 2002).
To establish a compatible interaction, biotrophic pathogens have to camouflage themselves against recognition, suppress the activation of plant defences or counter‐defend activated defences by the detoxification of potentially harmful compounds. Additionally, they have to redirect the host’s metabolic flow to their own benefit without killing the host (Panstruga, 2003; Glazebrook, 2005). Rust diseases of wheat are amongst the oldest plant diseases known to man. Early literature on wheat cultivation mentions these devastating diseases and their ability to destroy entire wheat crops. Numerous studies have been conducted on the life cycle of rust pathogens and their management (Marsalis and Goldberg, 2006). Leaf rust on wheat (Triticum aestivum L.)
stem and yellow rust (Kolmer, 1996). Genetic resistance against rusts is the most economical and preferable method of reducing yield losses due to leaf rust infection and can be fully utilized by knowing the identity of resistance genes in commonly used parental germplasm and released cultivars.
During SAR or induced systemic resistance (ISR), distal parts of a plant receive a signal from an infected part (Scheel, 1998). This signal allows the distal part of the plant to activate its own defence mechanisms as a preventative strategy. In a similar way, two neighbouring plants could communicate so that the uninfected plant could activate its defences based on an airborne signal coming from an infected neighbour (Huang et al., 2005; Gómez and Stuefer, 2006).
Originally proposed as pheromonal sensitivity in red alders and willows (Rhoades, 1983) and further corroborated in a study using poplars and maples (Baldwin and Schultz, 1983), the idea that plants might warn each other about an imminent attack was quite exciting because of the clear advantage that forewarning would mean for plants. It would remove the window of vulnerability that plants would otherwise suffer due to the lag during an induced response.
An important point to consider is whether communication between plants must be seen as mutualistic. Perhaps neighbouring plants are merely opportunistically responding to volatile signals released by the wounded plants as indicators of imminent herbivore attack. However, the interplant signalling can be interpreted as a natural outcome of sensitivity to itself.
Based on this, the aim of this study was to investigate some of the early events occurring after the infection of wheat with leaf rust on molecular level. An attempt was first made to clone and identify putative genes involved in these early events in order to elucidate the early signalling events in this particular interaction. Secondly, a possible inter‐plant communication event between infected and uninfected plants was investigated.
2.1
I
NTRODUCTION
Plants are major targets for pathogenic microbes looking for a source of nutrition. A complex array of interactions between plants and microbes has thus evolved to reflect both the nutrient acquisition strategies of the microbes as well as the defence strategies of plants.
Penetration of fungi through the plant cell wall represents an Achilles heel for the plant. For biotrophic fungi, it initiates a transition from extra‐cellular to invasive growth. Modification of the plant cell wall was recognized as a potential resistance mechanism against infection (Young, 1926; Franceschi et al., 2005). The termination of fungal pathogenesis at the cell wall is commonly associated with cell wall thickenings and the formation of callosites in the paramural space (Matern et al., 1995; McLusky et al., 1999; Snyder and Nicholson, 1990). The formation of these cell wall appositions (CWA) is usually accompanied by the co‐ localized accumulation of phenolics and ROS (Bestwick et al., 1998; Nicholson and Hammerschmidt, 1992; Thordal‐Christensen et al., 1997) aiming to deter invasion by the pathogen.
Specific host‐pathogen interaction models describing induced defence responses in plants, have been influenced by the gene‐for‐gene concept reported by Flor (1956). In these specific host‐pathogen interactions, resistance to a particular pathogen is conditional to the presence of a specific Avr‐gene in the pathogen and a specific R‐gene in the plant host.
2.2 P
LANT
‐P
ATHOGEN
I
NTERACTION
The biotrophic lifestyle of fungi is described as deriving energy from living plant cells (Mendgen and Hahn, 2002). Most parasitic biotrophs like mildews, rusts and smuts withdraw nutrients from shoot tissue and have no alternative energy source (Schulze‐Lefert and Panstruga, 2003). A characteristic feature of many but not all biotrophic fungi is their ability to form a specialized infection structure, the haustorium. Formation of these intracellular fungal structures requires successful penetration of the host cell wall. This is a complex process that exposes the intruding fungus to cell wall‐associated defence
the haustorial membrane, an extra‐haustorial matrix and the host plasma membrane following the contours of the haustorial membrane (Schulze‐Lefert and Panstruga, 2003). To establish a compatible interaction, biotrophic pathogens have to camouflage themselves against recognition, suppress the activation of plant defences or counter‐defend activated defences by the detoxification of potentially harmful compounds. Additionally, they have to redirect the host’s metabolic flow to their own benefit without killing the host.
The most common response of resistant plants to the cellular invasion by fungal pathogens is a rapid cell death, which forms part of the HR. However, the HR is not an obligatory component of the plant’s defence response since there are pathosystems in which resistance does not depend on the manifestation of HR (Heath, 2000).
2.2.1 W
HEATL
EAFR
USTRust‐causing fungi are obligatory biotrophic plant pathogens. Economically they are important biological agents that render damage to wheat plants. Leaf rust on wheat (Triticum aestivum L.) is caused by Puccinia triticina Eriks. and is found wherever wheat is grown. It is the most regularly occurring of the three wheat rusts, namely leaf rust, stem rust and yellow rust. Wheat cultivars that are susceptible to leaf rust suffer from yield reductions of between 5 to 30% or more, depending on the stage of crop development when the initial rust infection occurs (Kolmer, 1996). Wheat rust fungi spread in the form of clonally produced dikaryotic urediniospores, which can be blown by the wind for thousands of kilometers from initial infection sites (Roelfs, 1989). Thus, epidemics of wheat rusts can occur on a continental scale because of the widespread dispersal of urediniospores.
Wheat rust fungi are highly specific obligate parasites that interact with wheat in a gene‐for‐ gene relationship (Person, 1959; Flor, 1971). This high degree of specificity has made durable rust resistance in wheat difficult to achieve, because the virulence of wheat rust fungi against wheat resistance genes is highly diverse, resulting in the existence of many different pathogenic races. Rust races that are virulent against cultivars containing
large geographic areas (Kolmer, 1999), thus rendering the resistance genes ineffective (Kolmer, 2005).
Genetic resistance is the most economical and preferred method of reducing yield losses due to leaf rust infection and can be fully utilized by knowing the identity of resistance genes in commonly used parental germplasm and released cultivars. Identification of leaf rust resistance genes allows for efficient incorporation of these genes into germplasm pools. Thus far, 52 different leaf rust resistance (Lr) genes conferring specific resistance to leaf rust, have been identified and assigned to specific chromosomes (Hiebert et al., 2005).
2.3 P
ATHOGEN
R
ECOGNITION
Plant responses to infection are usually initiated by the specific recognition of the pathogen and the transmission of the signal via plasma membrane‐bound receptors. A surveillance system of receptors, some of which are encoded by R‐genes, reacts by a similar mechanism to all classes of pathogens, irrespective of whether they are viruses, bacteria, fungi or nematodes (Di Gaspero and Cipriani, 2003).
Receptor‐mediated recognition at the site of infection initiates cellular and systemic signalling processes that activate multi‐component defence responses both at local and systemic levels, resulting in the rapid establishment of local resistance and delayed development of SAR (Scheel, 1998). The earliest reactions of plant cells include changes in plasma membrane permeability leading to calcium and proton influx (McDowell and Dangl, 2000). This in turn leads to the production of reactive oxygen intermediates (ROI) such as superoxide (O2‐) and hydrogen peroxide (H2O2) catalyzed by plasma membrane‐located NADPH oxidase and/or apoplastic peroxidases (Somssich and Hahlbrock, 1998).
These initial ion fluxes and production of ROI also trigger the localized production of secondary messengers for the initiation of HR and defence gene expression. Interactions between ROI, nitric oxide (NO) and salicylic acid (SA) have been postulated (Delledonne et al., 2001). Other interacting components might include specifically induced phospholipases which act on lipid‐bound unsaturated fatty acids within the membrane resulting in the release of linolenic acid (Creelman and Mullet, 1997). Linolenic acid in turn acts as substrate
(Hamberg and Gardner, 1992; Creelman and Mullet, 1997).
The expression of most of the inducible, defence‐related genes are regulated by signal pathways involving one or more of the three key regulators namely jasmonate (Bell and Mullet, 1991; Creelman et al., 1992), ethylene (Boller, 1995) and salicylic acid (Slaymaker et al., 2002; Takahashi et al., 2002; Kumar and Klessig, 2003; Van Wees and Glazebrook, 2003).
2.4 D
EFENCE
R
ESPONSES
Plants generally activate multiple defence responses upon pathogen attack, which leads to cellular reprogramming. The most effective defence response in plants is mediated by R‐ genes that are able to detect specific pathogenic races through recognition of pathogen encoded Avr proteins (Glazebrook, 1999).
2.4.1 S
YSTEMICR
ESISTANCEIn general, SAR may involve the activation of more than one biochemical pathway and is believed to be mediated by SA (Johal et al., 1995). The central role of SA as a signal transducer of SAR was demonstrated in transgenic plants where SA could not accumulate. These plants failed to express SAR (Gaffney et al., 1993). The formation of phenolic free‐ radicals, resulting from the interaction of SA with catalase or ascorbate peroxidases, has been proposed to be involved in the induction of SAR (Durner and Klessig, 1995). The discovery that the exogenous application of SA activate the same spectrum of disease resistance responses as the preliminary infection by a pathogen, led to the exploitation of SAR with the application of synthetic chemicals that act at, or just downstream, of SA but also producing a successful SAR (Gozzo, 2003). Correlating with the onset of SAR, plants express a set of PR‐proteins (Kessmann et al., 1994). Some of these PR proteins have been shown to have antimicrobial activity in vitro or to confer increased resistance when over‐ expressed in plants (Morrissey and Osbourn, 1999). Once established, SAR may last for a relatively long time (from weeks to months), during which any attempted invasion by certain pathogens is hampered.
effective against multiple pathogen types (Pieterse et al., 1998). ISR is independent of SA and is not associated with the activation of pathogenesis‐related (PR) protein 1 gene expression. Instead, ISR requires the signalling pathways responding to the plant growth hormones, JA and ethylene (Pieterse et al., 1998).
2.4.2 P
ROGRAMMEDC
ELLD
EATHCell death is an essential process in the plant’s life cycle (Vranová et al., 2002). Two modes of cell death have been described in plants: programmed cell death (PCD) and necrosis. PCD is controlled genetically and shares features characteristic of the apoptotic cell death in animal cells, including cell shrinkage, cytoplasmic and nuclear condensation, chromatin condensation and DNA fragmentation (O’Brien et al., 1998). Necrosis on the other hand results from severe and persistent trauma and is not considered to be genetically controlled (Vranová et al., 2002).
Programmed cell death can be controlled by small cytotoxic molecules, ROS such as H2O2 and O2‐ and lipid peroxidases (Jabs, 1999). The execution stage of cell death is associated with an uncontrolled production of ROS that overwhelms the normal protective mechanisms of cells (Palma and Kermode, 2003). Another possibility is that ROS production is simply a consequence of cells undergoing death and plays no role in the initiation or execution phases of cell death.
One form of PCD is the HR that appears to be an integral part of plant defence mechanisms against pathogens (Palma and Kermode, 2003). Fungal infection can trigger the HR in plants, a process characterized by the rapid death of plant cells immediately surrounding the site of infection, which effectively prevents the spread of the pathogen (Ivanov et al., 2005). The oxidative burst that precedes the HR is accompanied by the generation of ROS, mainly due to the activation of a plasma‐membrane associated NAD(P)H oxidase (Jabs, 1999).
ROIs and reactive nitrogen intermediates (RNIs) are highly toxic and may directly offer protection against the pathogen, but they are the most non‐discriminating defence molecules produced by the offended hosts (Veronese et al., 2003). In animals, they are produced and accumulate only in specific self‐sacrificing cells (Nathan and Shiloh, 2000).
in self‐sacrificing cells offers protection against the pathogen while limiting the damage to the host. ROIs and RNIs also participate in transcriptional reprogramming in and around the affected cell (Veronese et al., 2003).
Expression of genes encoding enzymes that detoxify ROS e.g. catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidases (APX), correlates with the induction of the HR and may protect neighbouring cells from the uncontrolled diffusion of ROS (del Rio et al., 1998; Heath, 2000).
2.4.3 H
2O
2Until recently, H2O2 was viewed mainly as a toxic cellular metabolite (Neill et al., 2002b). It is now clear that it can also function as a defence molecule that mediates responses to various stimuli in both plant and animal cells (Neill et al., 2002a).
H2O2 is continually generated from various sources during normal metabolism and a wide range of steady‐state H2O2 concentrations has been reported (Karpinski et al., 1999; Veljovic‐Jovanovic et al., 2001). It can also be generated by specific enzymes during an oxidative burst, which results in rapidly increasing H2O2 synthesis and release into the apoplast (Orozco‐Cárdenas, et al., 2001). This oxidative burst is a common response to pathogens, elicitors, wounding, heat, ultra‐violet light and ozone (Neill et al., 2002a). Knockout experiments demonstrated that the AtrbohD and AtrbohF genes encoding NADPH oxidase are required for H2O2 generation during fungal and bacterial challenges (Torres et al., 2002). H2O2 generated after pathogen attack mediates cross‐linking of cell wall proteins (Bradley et al., 1992) and plant cell wall‐bound phenolics (Grant and Loake, 2002). H2O2 also regulates the expression of various genes, including genes encoding antioxidants as well as genes leading to the production of H2O2. This indicates the complex way in which intracellular H2O2 concentrations are monitored and maintained at a constant level (Neill et al., 2002a). A microarray study showed that the expression of 1‐2% of all Arabidopsis genes was altered in H2O2‐treated cell cultures (Desikan et al., 2001). Amongst these, genes encoding antioxidant proteins and proteins potentially involved in PCD, as well as defence
2.4.4 S
ALICYLICA
CIDThe involvement of SA as a signal molecule in local defences and SAR has been extensively studied because of its ability to induce protection against pathogens (Slaymaker et al., 2002; Takahashi et al., 2002; Kumar and Klessig, 2003; Van Wees and Glazebrook, 2003). Increases in endogenous levels of SA and its conjugates in pathogen‐inoculated plants coincide with the elevated expression of genes encoding PR‐proteins and the activation of disease resistance (Morrissey and Osbourn, 1999). As a result, the plant becomes more resistant to pathogen attack.
Plants that cannot accumulate SA due to the presence of transgenes that encodes salicylic acid‐degrading enzymes, for example NahG, develop an HR response after a challenge with an avirulent pathogen, but do not exhibit systemic expression of defence genes and do not develop resistance to subsequent pathogen attacks (Glazebrook, 1999; Nawrath and Métraux, 1999; Van Wees and Glazebrook, 2003). In contrast, the exogenous application of SA or its synthetic functional analogue benzo(1,2,3)thiadiazole‐7‐carbothioic acid S‐methyl ester (BTH) results in the activation of PR‐gene expression and enhanced resistance to pathogens (Shah, 2003). The Arabidopsis thaliana non‐expressor of PR1 (NPR1) gene, also called non‐inducible immunity 1 (NIM1), is an important component of SA defence. Npr1 and nim1 mutant plants are insensitive to SA and this compromises their disease resistance (Cao et al., 1994; Delaney et al., 1995). An NPR1‐independent, SA‐mediated resistance mechanism also operates in Arabidopsis (Dong, 2001).
SA can specifically bind to a variety of plant proteins affecting their activity (Dempsey et al., 1999; Slaymaker et al., 2002). It can also activate gene expression by multiple mechanisms and at different steps in plant defence (Feys and Parker, 2000; Kunkel and Brooks, 2002).
2.4.5 J
ASMONICA
CIDThe ubiquitous presence of JA and its methyl ester in all higher plants examined so far suggests a prominent role for these molecules in plant metabolism (Mason et al., 1992). Jasmonates are derived from linolenic acid by a lipoxygenase (LOX)‐mediated oxygenation
from fatty acids through the action of LOX (Hamberg and Gardner, 1992).
In plants, jasmonates move easily in both the liquid and the vapour phases (Farmer and Ryan, 1990). Methyl jasmonate is especially volatile, suggesting that it might also act in the gaseous form in analogy to the plant hormone ethylene (Wasternack and Parthier, 1997). JA affect a variety of physiological processes, including root growth (Staswick et al., 1992), tuber formation (Koda, 1992), tendril coiling (Weiler et al., 1993), senescence of leaves and stomatal opening (Sembdner and Parthier, 1993). An additional role for jasmonates lies in the mediation of the plant responses to stresses such as pathogen and herbivore attack (Thaler et al., 2002).
Low concentrations of JA regulate proteinase inhibitors like thionin (Epple et al., 1995), osmotin (Xu et al., 1994) and proline‐rich cell wall protein (Creelman et al., 1992) at transcription level. It can also induce the accumulation of different enzymes involved in plant defence reactions such as chalcone synthase (Creelman et al., 1992), PAL (Gundlach et al., 1992) and LOX (Bell and Mullet, 1991). The induction of these proteins suggests a role for JA in helping plants to contain the growth of microorganisms.
Reports on the induction of a resistant state in plants by JA are contradictory. Some authors (Kogel et al., 1994; Schweizer et al., 1993) found no evidence for the induction of resistance by jasmonate in the barley‐Erysiphe graminis f.sp. hordei interaction, but Mitchell and Walters (1995) proved induced systemic protection in the same pathosystem by treating the first leaves of seedlings with methyl jasmonate.
2.4.6 E
THYLENEEthylene is a volatile plant hormone derived from methionine that is involved in numerous physiological processes (Kende, 1993). Ethylene is produced upon wounding or infection by pathogens, as well as by treatment with elicitors of defence responses (Boller, 1995). Exogenous application of ethylene to tobacco carrying the N gene for resistance to Tobacco Mosaic Virus (TMV) results in resistance to TMV marked by a decrease in the size of the necrosis (Van Loon and Antoniw, 1982).
Ethylene can induce the accumulation of some of the PR‐proteins such as β‐1,3‐glucanase and chitinase (Abeles et al., 1971; Yamamoto et al., 2004). Structural reinforcement of the cell wall through lignification and the accumulation of hydroxyproline‐rich cell wall proteins are also enhanced by ethylene (Boller, 1995). Although such results might suggest that ethylene is the signal involved in the induction of SAR, several experimental results indicate that ethylene might not be directly linked to the induction of SAR (Boller, 1995). The induction of chitinase and β‐1,3‐glucanase in pea pods by pathogens can take place in tissue treated with aminoethoxyvinylglycine (AVG), a potent inhibitor of ethylene biosynthesis. This indicates that ethylene synthesis after infection might be a symptom rather than a cause of the induction of defence reactions (Mauch et al., 1984).
2.5 C
HANGES IN
G
ENE
A
CTIVITY
Activation of signal transduction networks after pathogen recognition results in the reprogramming of cellular metabolism, involving large changes in gene transcriptional activity while basic incompatibility frequently results in the expression of defence related genes and localized host cell death (Yamamoto et al., 2004).
Plants contain many defence related proteins. In addition to R‐genes and genes encoding signal transduction proteins, they also possess downstream defence genes encoding PR‐ proteins (Van Loon, 1997), enzymes involved in the generation of phytoalexins (Flors and Nonell, 2006), enzymes involved in oxidative stress protection (May et al., 1998), lignification (Cano‐Delgado et al., 2003) and numerous others. Many of these genes are involved in the production of secondary metabolites such as those of the shikimate (Herrmann and Weaver, 1999) and phenylpropanoid pathways (Dixon et al., 2002).
2.5.1 M
OLECULARC
HAPERONESMolecular chaperones are key components contributing to cellular homeostasis in cells under both optimal and abnormal growth conditions (Grene, 2002). They are responsible for the folding, assembly, translocation and degradation of proteins in a broad array of cellular processes. They also function in the stabilization of proteins and membranes and can assist in protein refolding under stress conditions (Wang et al., 2004). Many molecular chaperones are stress proteins and most were originally identified as heat‐shock proteins
chaperones follow their early nomenclatures and are referred to as Hsps.
Except for the small Hsp (sHsp) family (Haslbeck, 2002), relatively little attention has been given to the role of the many other Hsps in plant responses to abiotic stress and the direct support for Hsp function during plant abiotic stress tolerance is limited. This, despite the fact that Hsps are known to be expressed in plants not only when they experience high temperature stress, but also in response to other environmental stresses such as water, salinity, osmotic, cold and oxidative stress (Boston et al., 1996; Bukau and Horwich, 1998). Hsp60, Hsp70 and Hsp90 interact with a wide variety of co‐chaperone proteins that regulate their activity or aid in the folding of specific substrate proteins (Bukau and Horwich, 1998). Hsp70 is also involved in the modulation of signal transducers such as protein kinase A, protein kinase C and protein phosphatase (Ding et al., 1998). Chaperones of the Hsp90 and Hsp70 families and their co‐chaperones were also found to interact with a growing number of signal molecules, including nuclear hormone receptors, tyrosine‐ and serine/threonine kinases, cell‐cycle and cell‐death regulators, demonstrating that they could play a key role in cellular signal transduction networks (Nollen and Morimoto, 2002).
In mammalian cells, the sHsps are known to be involved not only in protection against stress, but also in the modulation of other cellular functions such as apoptosis and differentiation via their participation in the regulation of cellular redox states (Arrigo, 2002). Although most of the studies were carried out in organisms other than plants, similar cross‐ talk mechanisms might operate in plants. For example, heat‐shock transcription‐factor‐ dependent expression of the antioxidant ascorbate peroxidases in Arabidopsis (Panchuk et al., 2002) suggests that heat‐shock factors (HSFs) might not only be involved in Hsp synthesis but also in oxidative stress regulation of antioxidant gene expression.
Mehlen et al. (1996) and Garrido et al. (1999) demonstrated an interaction between sHsps and glutathione in mammalian cells that resulted in increased resistance to cell death induced by the tumour necrosis factor or by hydrogen peroxide. Resistance was dependent on both the increase in reduced glutathione and increased expression of sHsps and was
in plant systems, but there is much evidence that suggests the importance of glutathione in the protection against ROS damage (May et al., 1998; Noctor et al., 1998).
The protective effects of Hsps can be attributed to the intra‐cellular network of the chaperone machinery in which many chaperones interact (Wang et al., 2004). During stress, many enzymes and structural proteins undergo functional changes. Therefore, maintaining proteins in their functional conformations, preventing aggregation of non‐native proteins, refolding of denatured proteins to regain their function and the removal of non‐functional, but potentially harmful polypeptides, are important for cell survival under stress.
Four distinct functions have been assigned to molecular chaperones (Grene, 2002). They can act as repair proteins, they can remove proteins that are irretrievably damaged and they can facilitate the import of newly synthesized proteins into the interior of organelles such as peroxisomes. The fourth function is as antioxidant molecules in conjunction with protein methionine‐sulfoxide reductase.
Many molecular chaperones described so far are members of the Hsp60 and Hsp70 families. The various functions of Hsp70s rely on their ability to bind to unfolded segments of proteins in an ATP‐dependent, reversible manner. All Hsp70s in prokaryotic and eukaryotic cells consist of an ATP‐binding domain and a peptide‐binding domain (PBD) (Hartl and Hayer‐Hartl, 2002). The two domains cooperate and in doing so Hsp70s undergo substantial conformational changes. It is generally agreed upon that the PBD of Hsp70 in its ATP form has an open binding pocket which recognizes unfolded segments of polypeptides (Bukau and Horwich, 1998; Mayer et al., 2001; Hartl and Hayer‐Hartl, 2002). Upon hydrolysis of ATP by Hsp70, the peptide‐binding pocket closes so that the ADP form holds the substrate tight. For their various functions Hsp70s require the cooperation with a set of co‐ chaperones which help in binding the polypeptide substrates and which support the ATP/ADP cycle (Bukau and Horwich, 1998; Kelley, 1998; Fan et al., 2003).
Induction of host Hsp synthesis in response to an encounter with a pathogen has at least two major causes. Firstly, infected cells are confronted with antimicrobial mechanisms which they have activated themselves against the pathogen during infection (Buchmeier and Heffron, 1990). Effective protection against its own effecter molecules (e.g. reactive radicals) becomes vital to the host for survival and hence the need for Hsps. Second, once
intracellular host metabolism and thus, not surprisingly, many of these pathogens are potent inducers of plant Hsp synthesis (Zügel and Kaufmann, 1999). As soon as a pathogen enters its host, it is confronted with several changes, some of which are very stressful. Inside the host a pathogen is usually confronted with ROI and RNI’s, attack by lysosomal enzymes and depletion of co‐factors. To protect itself against its host, the pathogen thus activates various evasion mechanisms including the synthesis of its own Hsps (Buchmeier and Heffron, 1990).
2.5.2 T
RANSCRIPTION FACTORSStress gene induction occurs primarily at the level of transcription and regulation of the expression patterns of specific stress related genes is an important part of the plant stress response (Rushton and Somssich, 1998). Plants devote larger portions of their genomes to genes encoding transcription factors compared to mammalian cells. The Arabidopsis genome, for example, contains more than 1500 transcription factor genes (Reichmann et al., 2000) and these transcription factors belong to large gene families, some of which are unique to plants.
Transcription factors can be defined as regulatory proteins that bind to short stretches of DNA in a sequence‐specific manner and mediate protein‐protein interactions (Després and Fobert, 2006). These interactions facilitate or interfere with the recruitment of RNA polymerase and the basal transcriptional machinery. Three of the largest families include the ethylene‐responsive‐element‐binding factors (ERF), basic‐domain leucine zipper (bZIP) and WRKY proteins which are implicated in several stress responses.
ERF proteins form a subfamily of the APETALA2 (AP2)/ethylene‐responsive‐element‐binding protein transcription factor family that is unique to plants. ERF proteins share a conserved 58‐59 amino acid domain (the ERF domain) that can bind to two similar cis‐elements: the GCC box, which is found in several PR gene promoters where it confers ethylene responsiveness and the C‐repeat (CRT)/dehydration‐responsive element motif, which is involved in the expression of dehydration‐ and low temperature responsive genes (Fujimoto
infection with different but overlapping kinetics and this may help to orchestrate the correct temporal defence response (Oñate‐Sánchez and Singh, 2002).
bZIPs form a large family of transcription factors in plants with 75 members in Arabidopsis (Jakoby et al., 2002). One class of bZIP proteins that is linked to stress responses comprises the TGA/octopine synthase (ocs)‐element‐binding factor proteins. These proteins bind to the activation sequence‐1 (as‐1)/ocs element which regulates the expression of PR1 and glutathione S‐transferase genes (Lebel et al., 1998; Chen and Singh, 1999). In Arabidopsis there are seven members of the TGA/OBF family which play roles in plant defence and development and have been shown to interact with the NPR1 protein of the SA‐dependent defence pathway (Zhang et al., 1999; Després et al., 2000; Niggeweg et al., 2000).
WRKY proteins form another family of transcription factors that are unique to plants (Eulgem et al., 2000). WRKY proteins contain either one or two WRKY domains – a 60 amino acid region that contains the amino acid sequence WRKYGQK or a zinc finger like motif. Specific WRKY family members show enhanced expression and/or DNA‐binding activity following induction by a range of pathogens, defence signals and wounding (Eulgem et al., 2000).
2.5.3 R‐
GENESDespite the great diversity in lifestyle and pathogenic mechanisms of disease‐causing organisms, R‐genes were found to encode proteins containing certain common motifs. The current classification of R‐genes recognizes four classes, coding for (1) cytoplasmic serine/threonine kinases e.g. the Pto gene in tomato (Tang et al., 1999); (2) proteins containing a nucleotide binding site (NBS) and leucine‐rich repeats (LRRs) together with either a Toll/Interleukin‐1 receptor (TIR) domain (e.g. the tobacco N gene (Liu et al., 2002)) or a coiled‐coil structure (CC) for example the Arabidopsis RPS2 gene (Bent et al., 1994); (3) extracellular LRRs anchored to a transmembrane domain (e.g. the tomato Cf‐9 gene (Jones et al., 1994)) and (4) receptor‐like protein kinases (RLKs) with extracellular LRRs and an intracellular serine/threonine kinase domain (e.g. the rice Xa21 (Song et al., 1995) and the wheat Lrk10 genes (Feuillet et al., 1997)).
event or with general elicitors produced as a result of the infection. Once the receiver domain is activated by ligand binding, either the NBS or the serine/threonine kinase domain becomes accessible to the downstream components of the signal transduction pathway, ultimately triggering the defence response (Bendahmane et al., 2002).
In addition to gene for gene recognition of a pathogen mediated by the R and Avr genes, nonhost resistance is achieved through communication of specific pathogen or plant cell wall derived signal molecules, called exogenous or endogenous elicitors respectively (Montesano et al., 2003). These elicitors are often low‐molecular‐weight compounds that are either synthesized or liberated from polymeric precursors during infection (Somssich and Hahlbrock, 1998). The chemical structure of these elicitors varies greatly and includes glycoproteins, peptides and oligosaccharides (Boudart et al., 1995; Montesano et al., 2003). Some proteinaceous elicitors are directly produced by bacterial or fungal pathogens, whereas biologically active oligosaccharides are released from pathogen and plant cell walls by hydrolases secreted by the two organisms. Complex and largely unresolved perception systems exist for these elicitors on the plant cell surface which activate multiple intracellular defence pathways (Odjakova and Hadjiivanova, 2001). The NBS‐LRR class is by far the largest group of resistance proteins (Bai et al., 2002). Two subgroups within the NBS‐LRR class have been recognized by the presence or absence of a TIR domain and structural similarity to the cytoplasmic defence domains of the Toll and interleukin‐1 receptor (Baker et al., 1997; Parker et al., 1997; Rock et al., 1998).
The first subgroup (TIR‐NBS‐LRR) includes N (tobacco mosaic virus resistance (Holmes, 1938)), L6 (flax rust resistance (Flor, 1947)), M (flax rust resistance (Anderson et al., 1997)) and RPP5 (downy mildew resistance (Parker et al., 1997)) which are all involved in defence processes. The second subgroup, which lacks the TIR region, includes the bacterial resistance proteins RPS2 (Bent et al., 1994), RPM1 (Grant et al., 1995), Xa1 (Yoshimura et al., 1998), Prf (Salmeron et al., 1996) and the fungal resistance protein I2 (Ori et al., 1997).
includes EDS1 (Parker et al., 1996) while proteins in the LZ class signal via a pathway that includes the NDR1 gene (Century et al., 1997).
Over the past few years, protein kinases have been identified for both race‐ and nonrace‐ specific elicitation of defence responses in plants. They often participate in the direct perception of elicitors of Avr products (Song et al., 1995; Cervone et al., 1997; Feuillet et al., 1997; Thomas et al., 1997), mediate defence required for the production of defence mechanisms and function as regulators of defence responses (Romeis, 2001). The phosphorylation of proteins, probably initiated by a receptor, is thought to relay the defence signal to different downstream effectors (Ligterink et al., 1997). In some cases, the receptor contains a kinase domain that may trigger the phosphorylation cascade whereas in others a secondary messenger such as Ca2+ may trigger the protein kinases (Blumwald et al., 1998).
2.6 I
NTERPLANT
C
OMMUNICATION
SAR is the process whereby distal parts of a plant receive a signal from an infected part (Scheel, 1998). This signal allows the distal part to activate its own defence mechanisms as a preventative defence strategy. In a similar way two neighbouring plants could communicate so that the uninfected plant could activate its defences based on an airborne signal coming from an infected neighbour. Intra‐plant communication and signal transduction have been described often, so the possibility of communication and signal transduction between plants, exists. If there are receptor proteins to facilitate intra‐plant communication, there should also be ones able to accept signals from other plants in the same area.
Plant communication is a loaded term that has come to encompass a broad definition. Most would accept a definition with the requirement that information can be exchanged, regardless of intent or fitness consequence for either party (Baldwin et al., 2002).
Studies on plant‐to‐plant communication are often received with scepticism. The major issues raised are as follows: (1) data suffers from statistical flaws such as pseudo replication, (2) the dose of chemicals applied in experiments is unrealistically high, (3) the mechanism is
realistic field conditions are lacking (Karban et al., 2000; Dicke and Bruin, 2001).
Plants have developed a multitude of inducible defence mechanisms against aggressive biotic agents. Defensive actions by plants induced via specific signal transduction events may negatively affect a herbivore’s physiology. An example is the accumulation of protease inhibitors in potato and soybean plants (Koiwa et al., 1997). Plants may also indirectly defend themselves against herbivores by emitting specific blends of volatiles that attract natural carnivorous enemies of herbivores (Dicke et al., 1990; Turlings et al., 1990; Takabayashi and Dicke, 1996; Arimura et al., 2001). In some cases these compounds are released when feeding ruptures pre‐existing internal or external secretory structures in which volatiles are synthesized and stored, while in other cases these volatiles are formed at the moment of damage (Gang et al., 2001).
After herbivore attack, plants release complex bouquets of volatiles into the air from their vegetative tissues. Predators and parasitoids of insect herbivores are attracted to herbivore‐induced volatile releases, showing a powerful indirect defence for plants (Baldwin et al., 2002). There is a broad diversity of known inducible volatiles, including alkenes, alkanes (Preston et al., 2001), two jasmonates (cis‐jasmone (Preston et al., 2001) and methyl jasmonate (Farmer and Ryan, 1990)) and methyl salicylate (MeSA) (Shulaev et al., 1997) but the dominating compounds tend to be terpenes (Arimura et al., 2001) and C6 green leaf volatiles (GLVs) (Holopainen, 2004; Van den Boom et al., 2004).
Inducible volatiles can be divided into two classes. The first class, the GLVs is released immediately (0‐5 min) after mechanical damage to leaves (Fall et al., 1999) while the second class, which consists mainly of terpenes, is synthesized after damage but only released a few hours after the initial damage took place (Dudareva et al., 2004). The compounds are also released from damaged and undamaged leaves (Holopainen, 2004).
Of all these proposed signals, only methyl jasmonate is detectable in volatile collections from sagebrush, where the chemical induced the accumulation of proteinase inhibitor 1 to higher levels than what could be induced by wounding (Karban et al., 2000). It was also
However, methyl jasmonate is released by sagebrush irrespective of damage, so receiver plants need to distinguish the signal from ‘background noise’. Karban et al. (2000) and Preston et al. (2001) found that sagebrush increase methyl jasmonate production by up to 16 times and can also change the isomeric conformation of methyl jasmonate to the biologically more active cis isomer. It was found that the trans:cis methyl jasmonate ratio changes from approximately 80:20 in undamaged plants to approximately 40:60 in damaged plants. It is thus hypothesized that the receiving plants use the more active cis isomer as an indicator of damage (Preston et al., 2001).
Methyl jasmonate is a biosynthetic product of the lipoxygenase or octadecanoid pathway, which can be induced under stress caused by herbivory (Bate and Rothstein, 1998). Jasmonic acid and methyl jasmonate are known to induce various aspects of biochemically based defences within the plant or in tissue cultures, but the volatility of the methyl ester potentiates aerial activity (Pickett and Poppy, 2001). When the plant is damaged by herbivory or simulated herbivory, methyl epi‐jasmonate is predominantly released and this has greater activity on recipient wild tobacco plants (Pickett and Poppy, 2001; Karban, 2001).
Ethylene emissions from lima bean leaves infested with spider mites have been observed and was reported by Xu et al. (1994) to activate some defence genes. Ethylene is thus also thought to be one of the candidate airborne signals involved in plant‐plant communication (Arimura et al., 2001).
Maize seedlings damaged by beet armyworm caterpillars release a specific cocktail of volatile terpenoids and indole that is recognized by parasitic wasps (Turlings et al., 1991). Volicitin (N‐(17‐hydroxylinolenoyl)‐L‐glutamine) present in the saliva of beet armyworm caterpillars has been identified as the major active elicitor for the formation of volatiles in maize (Alborn et al., 1997). Recently two genes, Igl and Stc1, whose expression is specifically induced by volicitin, have been isolated from maize. Igl encodes an indole‐3‐ glycerol‐phosphate lyase (IGL) (Frey et al., 2000) and Stc1 encodes a sesquiterpene cyclase (Shen et al., 2000). IGL cleaves indole‐3‐glycerol‐phosphate to form indole and glyceraldehyde‐3‐phosphate (Frey et al., 2000). Indole in turn is further metabolized to form two benzoxazinoids namely 2,4‐dihydroxy‐2H‐1,4‐benzoxazin‐3(4H)‐one (DIBOA) and it’s
Benzoxazinoids are natural pesticides found predominantly in the Poaceae and serve as important factors in host‐plant resistance to microbial diseases and insects (Gierl and Frey, 2001).
Emission of volatiles from pathogen‐infected plants may serve as a direct defence against pathogen infections. Several lipid‐derived volatiles, including (Z)‐3‐hexenol and (E)‐2‐ hexenal are released from Phaseolus vulgaris leaves during an HR response to Pseudomonas syringae pv. phaseolicola (Croft et al., 1993). Both (Z)‐3‐hexenol and (E)‐2‐hexenal are bacteriocidal but at different concentrations (Croft et al., 1993). Maize‐derived volatile compounds, hexanal and octanal, strongly inhibit growth of the fungus Aspergillus parasiticus on culture media (Zeringue et al., 1996). Peanut plants infected with white mould, Sclerotium rolfsii, emitted a mixture of lipoxygenase products, terpenoids, indole and MeSA which were both quantitatively and qualitatively different from volatiles collected from healthy plants (Huang et al., 2003). Among these volatiles, (Z)‐3‐hexenyl acetate, linalool and MeSA significantly inhibited fungal growth on solid culture media.
While some studies found no evidence for the transfer of information between damaged and undamaged plants (Preston et al., 2001), many others presented evidence supporting the hypothesis of information exchange between damaged and undamaged plants (Dicke et al., 1990; Karban et al., 2000; Arimura et al., 2001). An important question is whether information exchange between damaged and undamaged plants can be expected in all plant species. If plants of a certain species show the ability, the question is whether individuals of that species should always respond to information from damaged neighbours (Dicke and Bruin, 2001). Once evidence for plant‐to‐plant communication has been found, it becomes feasible to investigate to what extent plants are informed about local conditions and what strategies they can follow (Karban et al., 1999).
2.7 C
ONCLUDING
R
EMARKS
Like animals, plants have evolved an elaborate system to protect them against pathogen infections. Numerous attempts have been made in the past to identify and clone the
successful. The challenge now is to identify all the interacting mechanisms and to clarify the role of each in the defence response. The aim of this study was to investigate, on molecular level, some early events following the infection of wheat with leaf rust. An attempt was made to identify putative genes involved in these early events. Once these genes were identified, their relevance in the biochemical defence response was investigated.
Chapter 3
Cloning of Differentially Expressed cDNA
Fragments from Leaf Rust‐Infected Wheat
